Single domain antibodies capable of modulating bace activity

ABSTRACT

Described are single domain antibodies with a specificity for BACE1. More specifically, described are single variable-domain antibodies derived from camelids that bind to BACE1 and are capable of inhibiting the activity of BACE1. The antibodies can be used for research and medical applications. Specific applications include the use of BACE1-specific antibodies for the treatment of Alzheimer&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/736,389, filed Dec. 27, 2010, pending, which application is anational stage filing under 35 U.S.C. §371 of International ApplicationNo. PCT/EP2009/053985, filed on Apr. 3, 2009, and published in Englishon Oct. 8, 2009 as WO 2009/121948 A2, which application claims thebenefit of U.S. Patent Application Ser. No. 61/041,965, filed Apr. 3,2008.

TECHNICAL FIELD

Described are single domain antibodies with a specificity for BACE1.More specifically, described are single variable-domain antibodiesderived from camelids that bind to BACE1 and are capable of inhibitingthe activity of BACE1. The antibodies can be used for research andmedical applications. Specific applications include the use ofBACE1-specific antibodies for the treatment of Alzheimer's disease.

BACKGROUND

Alzheimer's disease (“AD”) is a devastating neurodegenerative diseasethat affects millions of elderly patients worldwide and is the mostcommon cause of nursing home admittance. AD is clinically characterizedby progressive loss of memory, orientation, cognitive function, judgmentand emotional stability. With increasing age, the risk of developing ADincreases exponentially, so that by age 85, some 20% to 40% of thepopulation is affected. Memory and cognitive function deterioraterapidly within the first five years after diagnosis of mild to moderateimpairment, and death due to disease complications is an inevitableoutcome. Definitive diagnosis of AD can only be made post-mortem, basedon histopathological examination of brain tissue from the patient. Twohistological hallmarks of AD are the occurrence of neurofibrillartangles of hyperphosphorylated tau protein and of proteinaceous amyloidplaques, both within the cerebral cortex of AD patients. The amyloidplaques are composed mainly of a peptide of 37 to 43 amino acidsdesignated “beta-amyloid,” also referred to as “beta amyloid,” “amyloidbeta,” “Aβ” or “Abeta.” It is now clear that the Abeta peptide isderived from a type 1 integral membrane protein, termed “beta amyloidprecursor protein” (also referred to as “APP”) through two sequentialproteolytic events. First, the APP is hydrolyzed at a site N-terminal ofthe transmembrane alpha helix by a specific proteolytic enzyme referredto as “beta-secretase” (the membrane-bound protease BACE1). The solubleN-terminal product of this cleavage event diffuses away from themembrane, leaving behind the membrane-associated C-terminal cleavageproduct, referred to as “C99.” The protein C99 is then furtherhydrolyzed within the transmembrane alpha helix by a specificproteolytic enzyme referred to as “gamma-secretase.” This secondcleavage event liberates the Abeta peptide and leaves amembrane-associated “stub.” The Abeta peptide thus generated is secretedfrom the cell into the extracellular matrix where it eventually formsthe amyloid plaques associated with AD.

Despite intensive research during the last 100 years, prognosis of ADpatients now is still quite the same as that of patients a century ago,since there is still no real cure available. There are two types ofdrugs approved by the U.S. Food and Drug Administration and used inclinics today to treat AD: Acetylcholinesterase (AchE) inhibitors andMemantine. There is ample evidence in the art that the amyloid betapeptide, the main component of the amyloid plaques that are specific tothe AD etiology, has a key role in the development of AD disease.Therefore, one of the most favorite strategies to lower Aβ is todiminish its production by γ- and β-secretase inhibitors. One strategywas the development of gamma-secretase inhibitors; however, suchinhibitors often result in serious side effects since gamma-secretase isinvolved in the proteolytic processing of at least 30 proteins.

Yet another attractive strategy is the development of BACE1 inhibitors.BACE1 is produced as a prepropeptide. The 21-amino acid signal peptidetranslocates the protease into the ER where the signal peptide iscleaved off and from where BACE1 is then directed to the cell surface.After its passage through the trans-Golgi network (TGN), part of BACE1is targeted to the cell surface from where it is internalized into earlyendosomal compartments. BACE1 then either enters a direct recyclingroute to the cell surface or is targeted to late endosomal vesiclesdestined for the lysosomes or for the TGN. At the TGN, it might beretransported to the cell membrane. Given its long half-life and fastrecycling rate, mature BACE1 may cycle multiple times between cellsurface, endosomal system and TGN during the course of its lifespan.BACE1 inhibitory antibodies are described in US20060034848.

SUMMARY OF THE DISCLOSURE

Herein, we sought to develop alternative inhibitors of the activity ofBACE1 through the generation of single chain antibodies with aspecificity for BACE1. In the resulting collection of binders of BACE1,we identified inhibitors of BACE1. In particular, these BACE1-specificcamelid antibodies capable of inhibiting BACE1 activity can be used forthe treatment of Alzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Amino acid sequence alignment of the variable domain of theBACE1-specific dromedary HCAbs (listed, in order, Nb_B1, Nb_B2, Nb_B3,Nb_B5, Nb_B8, Nb_B9, Nb_B10, Nb_B11, Nb_B12, Nb_B15, Nb_B16, Nb_B21,Nb_B25, and Nb_B26, corresponding with SEQ ID NOS:1-14, respectively;and Nb_B4, Nb_B6, Nb_B7, Nb_B13, Nb_B14 and Nb_B24, corresponding withSEQ ID NOS:38-43, respectively). V_(H)H hallmark residues (F/Y₃₇, E/Q₄₄,R₄₅ and G₄₇) are indicated in bold, whereas residues characteristic fora VH-motif (L₁₁, V₃₇, G₄₄, L₄₅ and W₄₇) are labeled in italics. Cysteineresidues other than the canonical C₂₂ and C₉₂ are underlined. Numberingand grouping of residues into either framework or CDR regions are asdefined by Kabat (Kabat et al., 1991).

FIG. 2: Capacity of the different BACE1 binders to recognize theirantigen. Panel A: A RaPID plot representing the kinetic rate valuesk_(on) (M⁻¹s⁻¹) and k_(off) (s⁻¹) for the NANOBODY®-immunogeninteractions as determined by surface plasmon resonance (BIAcore). Theratio of k_(off) to k_(on) gives the dissociation constant or K_(D).Kinetic constants were measured at pH 7.0 (black spots) and pH 5.0 (grayspots). The majority of the BACE1 binders has K_(D) values between 10 nMand 100 nM at both pH conditions. Panel B: Capacity of the differentBACE1 binders to pull down BACE1 from cell lysates. Lysates ofBACE1-transfected COS cells were incubated with equal amounts (2 μg) ofthe various recombinant his-tagged anti-BACE1 NANOBODIES®. Followingpull down of the NANOBODIES®, samples were subjected to SDS-PAGE andanalyzed by Western blotting using anti-BACE1 (ProSci). Nb_BCIILP andNb_Aβ3, raised against beta-lactamase BCII 569/H (Conrath et al., 2001a)and Aβ peptide, respectively, were used as negative controls. Only partof the NANOBODIES®, raised against non-glycosylated BACE1 ectodomain,are able to efficiently capture glycosylated BACE1 from COS celllysates.

FIG. 3: Effect of Nb_B26 and Nb_B9 on APP processing in cells. Panel A:Schematic representation of the cDNA construct used to express V_(H)Hsinto mammalian cells. The construct consists of the V_(H)H cDNA, fusedat its C-terminus to the signal peptide of BACE1, to ensure ERtranslocation, and at its C-terminus it is fused to a myc-epitope tag.Panel B: Nb_B9, but not Nb_B26, adversely affects β-site APP processingupon transient overexpression. COS-B1 cells, stably expressing lowlevels of BACE1, were co-transfected with APP_(Sw) and either Nb_B26 orNb_B9. Control cells were either transfected with empty vector or withAPP_(Sw) alone. Two days after transfection, cells were lysed and totalprotein extracts were analyzed by Western blotting using anti-myc,anti-BACE1 (ProSci) and B63.1, to detect V_(H)Hs, BACE1 and APP fulllength and CTFs (C83 and C99), respectively. One representativeexperiment is shown. Panel C: Western blots as the one shown in Panel Cwere probed with GARIR800, an infrared-coupled secondary antibody, andthen scanned on an ODYSSEY® scanner. The signal intensity of the APPCTFs was quantified using the ODYSSEY® Application Software v1.2.15(LI-COR). The ratio of β-CTF to total CTFs (mean±SEM, n=8 to 10),normalized to the ratio of non-transfected cells (set as 1), shows thatNb_B9 could consistently decrease activity by about 30% (t-test,p<0.001), whereas Nb_B26 had no impact on APP processing.

FIG. 4: V_(H)H Nb_B9 inhibits β-secretase cleavage of APP by adding tothe medium of cultured cells. Neuroblastoma cells SH-SY5Y/APPwt weretreated with 3 μM NANOBODIES® for 24 hours, sAPPα and sAPPIβ fromconditioned medium were analyzed by Western blot. Cells treated withNANOBODY® B9 (SEQ ID NO:6) showed a significant decrease in sAPPIβproducing.

FIG. 5: Amino acid sequence alignment of BACE1-specific V_(H)Hs isolatedfrom dromedary and llama V_(H)H libraries (SEQ ID NOS:6, and 15-28).Numbering and grouping of residues into either framework or CDR regionsare as defined by Kabat (Kabat et al., 1991).

FIG. 6: Western blot analysis of sAPPIβ and sAPPα from conditionedmedium of neuroblastoma cells SH-SY5Y/APPwt treated with NANOBODIES® byadding to the medium at final concentration of 20 μM. Cells treated withNANOBODY® B9 (SEQ ID NO:6), 10C4 (SEQ ID NO:22), and 4A2 (SEQ ID NO:26)showed a significant decrease in sAPPIβ producing.

FIG. 7: The inhibition effects of different NANOBODIES® (10 μM) on BACE1activity in FRET assay at a concentration of 10 μM. In this cell-freeenzymatic assay, Nb_B9 (SEQ ID NO:6), Nb_10C4 (SEQ ID NO:22), Nb_4A2(SEQ ID NO:26), and Nb_1B3 (SEQ ID NO:15) significantly modulate BACE1activity.

FIG. 8: Dose-response curve of NANOBODIES® 10C4 (Panel A), 4A2 (PanelB), and B9 (Panel C) on BACE1 cleavage activity in FRET assay using asmall peptide substrate. Nb_10C4 and Nb_4A2 significantly inhibit BACE1activity. Nb_B9 significantly increases BACE1 activity.

FIG. 9: Dose-response curve of Nb_B9 on BACE1 cleavage activity inMBP-ELISA using a big peptide substrate. In this cell-free enzymaticassay, Nb_B9 significantly inhibits BACE1 activity.

FIG. 10: V_(H)Hs Nb_B9 (SEQ ID NO:6) and Nb_4A2 (SEQ ID NO:26) inhibitBACE1 cleavage of APPwt in primary cultured mouse neurons, as reflectedin a decrease of Aβ, sAPPβ and CTFβ. Primary cultured neurons fromwild-type mice were transduced with APPwt by Semliki Forest Virus (SFV),and then treated with purified Nb_B9 and Nb_4A2 by adding to the mediumat a final concentration of 20 μM (V_(H)Hs were first dissolved in PBS),neurons treated with PBS were used as a negative control. After a16-hour treatment, conditioned medium and cell extract were analyzed byWestern blot for APP-FL, CTFβ, CTFα, Aβ, sAPPβ and sAPPα/β.

FIG. 11: Dose response curve of V_(H)Hs Nb_B9 (SEQ ID NO:6) inhibitingBACE1 in primary cultured mouse neurons established by metaboliclabeling assays after a 6-hour treatment. Primary cultured neurons fromwild-type mice were transduced with APPwt by Semliki Forest Virus (SFV),and treated with purified V_(H)H B9 by adding to the medium serialdilutions (V_(H)H B9 was first dissolved and diluted in PBS). Neuroncultures were metabolic labeled for 6 hours, APP-FL and CTFβ from cellextracts were analyzed by phosphorimaging, while sAPPβ, Aβ and sAPPαfrom conditioned medium were analyzed by Western blot.

DETAILED DESCRIPTION

Described are BACE1 single variable-domain antibodies that can be usedin research and medical applications. More specifically, described isthe detection of BACE1 overexpression and to the treatment ofAlzheimer's disease using BACE1 single domain antibodies. As usedherein, the antibodies are devoid of any light chain but comprise atleast one heavy chain antibody. In a particular embodiment, the variabledomain of a heavy chain antibody is derived from camelids. Such avariable domain heavy chain antibody is herein designated as a NANOBODY®or a V_(H)H antibody. NANOBODY®, NANOBODIES®, and NANOCLONE® aretrademarks of Ablynx NV (Belgium).

Thus, in a first embodiment, provided is a single variable-domainantibody, devoid of a light chain, specifically binding to BACE1. In aparticular embodiment, the single domain antibody is derived fromcamelids. In the family of “camelids,” immunoglobulins devoid of lightpolypeptide chains are found. “Camelids” comprise old-world camelids(Camelus bactrianus and Camelus dromaderius) and new world camelids (forexample, Lama paccos, Lama glama and Lama vicugna).

In another embodiment, provided is a single domain antibody derived fromcamelids, which amino acid sequence comprises SEQ ID NOS:1-28. The aminoacid sequences of the dromedary/llama NANOBODIES® (also designated asV_(H)H antibodies) are depicted in FIGS. 1 and 5. NANOBODY® B1 (Nb_B1)corresponds with SEQ ID NO:1, NANOBODY® B2 (Nb_B2) corresponds with SEQID NO:2, NANOBODY® B3 (Nb_B3) corresponds with SEQ ID NO:3, NANOBODY® B5(Nb_B5) corresponds with SEQ ID NO:4, NANOBODY® B8 (Nb_B8) correspondswith SEQ ID NO:5, NANOBODY® B9 (Nb_B9) corresponds with SEQ ID NO:6,NANOBODY® B10 (Nb_B10) corresponds with SEQ ID NO:7, NANOBODY® 11(Nb_B11) corresponds with SEQ ID NO:8, NANOBODY® 12 (Nb_B12) correspondswith SEQ ID NO:9, NANOBODY® 15 (Nb_B15) corresponds with SEQ ID NO:10,NANOBODY® 16 (Nb_B16) corresponds with SEQ ID NO:11, NANOBODY® 21(Nb_B21) corresponds with SEQ ID NO:12, NANOBODY® 25 (Nb_B25)corresponds with SEQ ID NO:13, NANOBODY® 26 (Nb_B26) corresponds withSEQ ID NO:14, NANOBODY® 1B3 (Nb_1B3) corresponds with SEQ ID NO:15,NANOBODY® 10C2 (Nb_10C2) corresponds with SEQ ID NO:16, NANOBODY® 12B6(Nb_12B6) corresponds with SEQ ID NO:17, NANOBODY® 10B5 (Nb_10B5)corresponds with SEQ ID NO:18, NANOBODY® 13A5 (Nb_13A5) corresponds withSEQ ID NO:19, NANOBODY® 2C6 (Nb2C6) corresponds with SEQ ID NO:20,NANOBODY® 6A4 (Nb_6A4) corresponds with SEQ ID NO:21, NANOBODY® 10C4(Nb_10C4) corresponds with SEQ ID NO:22, NANOBODY® 13B6 (Nb_13B6)corresponds with SEQ ID NO:23, NANOBODY® 1A4 (Nb_1A4) corresponds withSEQ ID NO:24, NANOBODY® 2B6 (Nb_2B6) corresponds with SEQ ID NO:25,NANOBODY® 4A2 (Nb_4A2) corresponds with SEQ ID NO:26, NANOBODY® 1D4(Nb_1D4) corresponds with SEQ ID NO:27 and NANOBODY® 9D3 (Nb_9D3)corresponds with SEQ ID NO:28.

In yet another embodiment, the single domain antibody is capable ofinhibiting the activity of BACE1. It is understood that “inhibition ofthe activity” is equivalent with the wording “down-regulating theactivity.” Generally, “inhibition” means that the activity of BACE1 isinhibited by at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95% or even 96%, 97%,98%, 99% or even 100%. Inhibition of BACE1 can be determined asmentioned herein further in the examples.

In yet another embodiment, the single domain antibody is capable ofinhibiting the activity of BACE1 and it comprises at least one of thecomplementarity-determining regions (CDRs) with an amino acid sequenceselected from the group comprising SEQ ID NOS:29-37.

In yet another embodiment, the single domain antibody is capable ofpreventing the uptake of pro-BACE1 and its amino acid sequence comprisesSEQ ID NOS:6, 22 or 26.

It should be noted that the term “NANOBODY®” as used herein in itsbroadest sense is not limited to a specific biological source or to aspecific method of preparation. For example, the NANOBODIES® hereof cangenerally be obtained: (1) by isolating the V_(H)H domain of a naturallyoccurring heavy chain antibody; (2) by expression of a nucleotidesequence encoding a naturally occurring V_(H)H domain; (3) by“humanization” of a naturally occurring V_(H)H domain or by expressionof a nucleic acid encoding a such humanized V_(H)H domain; (4) by“camelization” of a naturally occurring VH domain from any animalspecies and, in particular, from a mammalian species, such as from ahuman being, or by expression of a nucleic acid encoding such acamelized VH domain; (5) by “camelization” of a “domain antibody” or“Dab” as described in the art, or by expression of a nucleic acidencoding such a camelized VH domain; (6) by using synthetic orsemi-synthetic techniques for preparing proteins, polypeptides or otheramino acid sequences known per se; (7) by preparing a nucleic acidencoding a NANOBODY® using techniques for nucleic acid synthesis knownper se, followed by expression of the nucleic acid thus obtained; and/or(8) by any combination of one or more of the foregoing. One preferredclass of NANOBODIES® corresponds to the V_(H)H domains of naturallyoccurring heavy chain antibodies directed against BACE1. As furtherdescribed herein, such V_(H)H sequences can generally be generated orobtained by suitably immunizing a species of camelid with BACE1 (i.e.,so as to raise an immune response and/or heavy chain antibodies directedagainst BACE1), by obtaining a suitable biological sample from thecamelid (such as a blood sample, serum sample or sample of B-cells), andby generating V_(H)H sequences directed against BACE1, starting from thesample, using any suitable technique known per se. Such techniques willbe clear to the skilled person.

Alternatively, such naturally occurring V_(H)H domains against BACE1 canbe obtained from naive libraries of Camelid V_(H)H sequences, forexample, by screening such a library using BACE1 or at least one part,fragment, antigenic determinant or epitope thereof using one or moreknown screening techniques per se. Such libraries and techniques are,for example, described in WO9937681, WO0190190, WO03025020 andWO03035694. Alternatively, improved synthetic or semi-syntheticlibraries derived from naive V_(H)H libraries may be used, such asV_(H)H libraries obtained from naive V_(H)H libraries by techniques suchas random mutagenesis and/or CDR shuffling, such as, for example,described in WO0043507. Yet another technique for obtaining V_(H)Hsequences directed against BACE1 involves suitably immunizing atransgenic mammal that is capable of expressing heavy chain antibodies(i.e., so as to raise an immune response and/or heavy chain antibodiesdirected against BACE1), obtaining a suitable biological sample from thetransgenic mammal (such as a blood sample, serum sample or sample ofB-cells), and then generating V_(H)H sequences directed against BACE1starting from the sample, using any suitable technique known per se. Forexample, for this purpose, the heavy chain antibody-expressing mice andthe further methods and techniques described in WO02085945 and inWO04049794 can be used.

A particularly preferred class of NANOBODIES® hereof comprisesNANOBODIES® with an amino acid sequence that corresponds to the aminoacid sequence of a naturally occurring V_(H)H domain, but that has been“humanized,” i.e., by replacing one or more amino acid residues in theamino acid sequence of the naturally occurring V_(H)H sequence (and, inparticular, in the framework sequences) by one or more of the amino acidresidues that occur at the corresponding position(s) in a VH domain froma conventional four-chain antibody from a human being. This can beperformed in a manner known per se, which will be clear to the skilledperson, for example, on the basis of the further description herein andthe prior art on humanization referred to herein. Again, it should benoted that such humanized NANOBODIES® of the invention can be obtainedin any suitable manner known per se (i.e., as indicated under points(1)-(8) above) and, thus, are not strictly limited to polypeptides thathave been obtained using a polypeptide that comprises a naturallyoccurring V_(H)H domain as a starting material.

Another particularly preferred class of NANOBODIES® of the inventioncomprises NANOBODIES® with an amino acid sequence that corresponds tothe amino acid sequence of a naturally occurring VH domain, but that hasbeen “camelized,” i.e., by replacing one or more amino acid residues inthe amino acid sequence of a naturally occurring VH domain from aconventional four-chain antibody by one or more of the amino acidresidues that occur at the corresponding position(s) in a V_(H)H domainof a heavy chain antibody. Such “camelizing” substitutions arepreferably inserted at amino acid positions that form and/or are presentat the VH-VL interface, and/or at the so-called Camelidae hallmarkresidues, as defined herein (see, for example, WO9404678). Preferably,the VH sequence that is used as a starting material or starting pointfor generating or designing the camelized NANOBODY® is preferably a VHsequence from a mammal, more preferably, the VH sequence of a humanbeing, such as a VH3 sequence. However, it should be noted that suchcamelized NANOBODIES® of the invention can be obtained in any suitablemanner known per se (i.e., as indicated under points (1)-(8) above) and,thus, are not strictly limited to polypeptides that have been obtainedusing a polypeptide that comprises a naturally occurring VH domain as astarting material. For example, both “humanization” and “camelization”can be performed by providing a nucleotide sequence that encodes anaturally occurring V_(H)H domain or VH domain, respectively, and thenchanging, in a manner known per se, one or more codons in the nucleotidesequence in such a way that the new nucleotide sequence encodes a“humanized” or “camelized” NANOBODY® of the invention, respectively.This nucleic acid can then be expressed in a manner known per se, so asto provide the desired NANOBODY® of the invention.

Alternatively, based on the amino acid sequence of a naturally occurringV_(H)H domain or VH domain, respectively, the amino acid sequence of thedesired humanized or camelized NANOBODY® of the invention, respectively,can be designed and then synthesized de novo using techniques forpeptide synthesis known per se. Also, based on the amino acid sequenceor nucleotide sequence of a naturally occurring V_(H)H domain or VHdomain, respectively, a nucleotide sequence encoding the desiredhumanized or camelized NANOBODY® hereof, respectively, can be designedand then synthesized de novo using techniques for nucleic acid synthesisknown per se, after which the nucleic acid thus obtained can beexpressed in a manner known per se, so as to provide the desiredNANOBODY® of the invention. Other suitable methods and techniques forobtaining the NANOBODIES® hereof and/or nucleic acids encoding the same,starting from naturally occurring VH sequences or preferably V_(H)Hsequences, will be clear from the skilled person, and may, for example,comprise combining one or more parts of one or more naturally occurringVH sequences (such as one or more FR sequences and/or CDR sequences),one or more parts of one or more naturally occurring V_(H)H sequences(such as one or more FR sequences or CDR sequences), and/or one or moresynthetic or semi-synthetic sequences, in a suitable manner, so as toprovide a NANOBODY® hereof or a nucleotide sequence or nucleic acidencoding the same.

According to one non-limiting aspect hereof, a NANOBODY® may be asdefined herein, but with the proviso that it has at least “one aminoacid difference” (as defined herein) in at least one of the frameworkregions compared to the corresponding framework region of a naturallyoccurring human VH domain, and, in particular, compared to thecorresponding framework region of DP-47. More specifically, according toone non-limiting aspect of the invention, a NANOBODY® may be as definedherein, but with the proviso that it has at least “one amino aciddifference” (as defined herein) at at least one of the Hallmark residues(including those at positions 108, 103 and/or 45) compared to thecorresponding framework region of a naturally occurring human VH domain,and, in particular, compared to the corresponding framework region ofDP-47. Usually, a NANOBODY® will have at least one such amino aciddifference with a naturally occurring VH domain in at least one of FR2and/or FR4, and, in particular, at at least one of the Hallmark residuesin FR2 and/or FR4 (again, including those at positions 108, 103 and/or45). Also, a humanized NANOBODY® hereof may be as defined herein, butwith the proviso that it has at least “one amino acid difference” (asdefined herein) in at least one of the framework regions compared to thecorresponding framework region of a naturally occurring V_(H)H domain.More specifically, according to one non-limiting aspect hereof, aNANOBODY® may be as defined herein, but with the proviso that it has atleast “one amino acid difference” (as defined herein) at at least one ofthe Hallmark residues (including those at positions 108, 103 and/or 45)compared to the corresponding framework region of a naturally occurringV_(H)H domain. Usually, a NANOBODY® will have at least one such aminoacid difference with a naturally occurring V_(H)H domain in at least oneof FR2 and/or FR4, and, in particular, at at least one of the Hallmarkresidues in FR2 and/or FR4 (again, including those at positions 108, 103and/or 45). As will be clear from the disclosure herein, it is alsowithin the scope hereof to use natural or synthetic analogs, mutants,variants, alleles, homologs and orthologs (herein collectively referredto as “analogs”) of the NANOBODIES® hereof as defined herein, and, inparticular, analogs of the NANOBODIES® of SEQ ID NOS:6, 22 or 26. Thus,according to one embodiment, the term “NANOBODY® hereof” in its broadestsense also covers such analogs. Generally, in such analogs, one or moreamino acid residues may have been replaced, deleted and/or added,compared to the NANOBODIES® hereof as defined herein. Suchsubstitutions, insertions or deletions may be made in one or more of theframework regions and/or in one or more of the CDRs, and, in particular,analogs of the CDRs of the NANOBODIES® of SEQ ID NOS:6, 22 or 26, theCDRs corresponding with SEQ ID NOS:29-37 (see Table 1, FIGS. 1 and 5).

TABLE 1  CDRs of BACE1-specific NANOBODIES ® Nb CDR1 CDR2 CDR3 Nb_B9EYTYGYCSMG TITSDGSTSYV KTCANKLGAKFIS (SEQ ID (SEQ ID NO: DSVKG (SEQ(SEQ ID NO: NO: 6) 29) ID NO: 30) 31) Nb_10C4 GYTYSTCSMA SIRNDGSTAYARIGVGPGGTCSIYA (SEQ ID (SEQ ID NO: DSVKG (SEQ PY (SEQ ID NO: NO: 22) 32)ID NO: 33) 34) Nb_4A2 GFTFETQYMT SINSGGTIKYY GQWAGVGAASS (SEQ ID (SEQ ID NO: ANSSVKG (SEQ (SEQ ID NO: NO: 26) 35) ID NO: 36) 37)

When such substitutions, insertions or deletions are made in one or moreof the framework regions, they may be made at one or more of theHallmark residues and/or at one or more of the other positions in theframework residues. Substitutions, insertions or deletions at theHallmark residues are generally less preferred (unless these aresuitable humanizing substitutions as described herein). By means ofnon-limiting examples, a substitution may, for example, be aconservative substitution (as described herein) and/or an amino acidresidue may be replaced by another amino acid residue that naturallyoccurs at the same position in another V_(H)H domain. Thus, any one ormore substitutions, deletions or insertions, or any combination thereof,that either improve the properties of the NANOBODY® hereof or that atleast do not detract too much from the desired properties or from thebalance or combination of desired properties of the NANOBODY® of theinvention (i.e., to the extent that the NANOBODY® is no longer suitedfor its intended use) are included within the scope of the disclosure.

A skilled person will generally be able to determine and select suitablesubstitutions, deletions or insertions, or suitable combinationsthereof, based on the disclosure herein and optionally after a limiteddegree of routine experimentation, which may, for example, involveintroducing a limited number of possible substitutions and determiningtheir influence on the properties of the NANOBODIES® thus obtained. Forexample, and depending on the host organism used to express theNANOBODY® or polypeptide of the disclosure, such deletions and/orsubstitutions may be designed in such a way that one or more sites forpost-translational modification (such as one or more glycosylationsites) are removed, as will be within the ability of the person skilledin the art.

Alternatively, substitutions or insertions may be designed so as tointroduce one or more sites for attachment of functional groups (asdescribed herein), for example, to allow site-specific pegylation. Onepreferred class of analogs of the NANOBODIES® hereof compriseNANOBODIES® that have been humanized (i.e., compared to the sequence ofa naturally occurring NANOBODY® of the invention). As mentioned in thebackground art cited herein, such humanization generally involvesreplacing one or more amino acid residues in the sequence of a naturallyoccurring V_(H)H with the amino acid residues that occur at the sameposition in a human VH domain, such as a human VH3 domain.

Examples of possible humanizing substitutions or combinations ofhumanizing substitutions will be clear to the skilled person, from thepossible humanizing substitutions mentioned in the background art citedherein, and/or from a comparison between the sequence of a NANOBODY® andthe sequence of a naturally occurring human VH domain. The humanizingsubstitutions should be chosen such that the resulting humanizedNANOBODIES® still retain the favorable properties of NANOBODIES® asdefined herein and, more preferably, such that they are as described foranalogs in the preceding paragraphs. A skilled person will generally beable to determine and select suitable humanizing substitutions orsuitable combinations of humanizing substitutions, based on thedisclosure herein and optionally after a limited degree of routineexperimentation, which may, for example, involve introducing a limitednumber of possible humanizing substitutions and determining theirinfluence on the properties of the NANOBODIES® thus obtained. Generally,as a result of humanization, the NANOBODIES® of the disclosure maybecome more “human-like,” while still retaining the favorable propertiesof the NANOBODIES® of the invention as described herein. As a result,such humanized NANOBODIES® may have several advantages, such as areduced immunogenicity, compared to the corresponding naturallyoccurring V_(H)H domains.

Again, based on the disclosure herein and optionally after a limiteddegree of routine experimentation, the skilled person will be able toselect humanizing substitutions or suitable combinations of humanizingsubstitutions that optimize or achieve a desired or suitable balancebetween the favorable properties provided by the humanizingsubstitutions on the one hand and the favorable properties of naturallyoccurring V_(H)H domains on the other hand. Examples of suchmodifications, as well as examples of amino acid residues within theNANOBODY® sequence that can be modified in such a manner (i.e., eitheron the protein backbone but preferably on a side chain), methods andtechniques that can be used to introduce such modifications and thepotential uses and advantages of such modifications will be clear to theskilled person. For example, such a modification may involve theintroduction (e.g., by covalent linking or in another suitable manner)of one or more functional groups, residues or moieties into or onto theNANOBODY® of the invention, and, in particular, of one or morefunctional groups, residues or moieties that confer one or more desiredproperties or functionalities to the NANOBODY® of the invention.

Examples of such functional groups and of techniques for introducingthem will be clear to the skilled person, and can generally comprise allfunctional groups and techniques mentioned in the general background artcited hereinabove, as well as the functional groups and techniques knownper se for the modification of pharmaceutical proteins and, inparticular, for the modification of antibodies or antibody fragments(including ScFvs and single domain antibodies), for which reference is,for example, made to Remington's Pharmaceutical Sciences, 16th ed., MackPublishing Co., Easton, Pa. (1980). Such functional groups may, forexample, be linked directly (for example, covalently) to a NANOBODY® ofthe invention, or optionally, via a suitable linker or spacer, as willagain be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including, but not limited to,(single) domain antibodies and ScFvs); reference is made to, forexample, Chapman, Nat. Biotechnol. 54:531-545 (2002); by Veronese andHarris, Adv. Drug Deliv. Rev. 54:453-456 (2003); by Harris and Chess,Nat. Rev. Drug Discov. 2 (2003); and in WO04060965. Various reagents forpegylation of proteins are also commercially available, for example,from Nektar Therapeutics, USA. Preferably, site-directed pegylation isused, in particular, via a cysteine-residue (see, for example, Yang etal., Protein Engineering 16, 10:761-770 (2003). For example, for thispurpose, PEG may be attached to a cysteine residue that naturally occursin a NANOBODY® hereof. A NANOBODY® hereof may be modified so as tosuitably introduce one or more cysteine residues for attachment of PEG,or an amino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of aNANOBODY® hereof, all using techniques of protein engineering known perse to the skilled person. Preferably, for the NANOBODIES® and proteinsof the disclosure, a PEG is used with a molecular weight of more than5000, such as more than 10,000 and less than 200,000, such as less than100,000; for example, in the range of 20,000-80,000.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the NANOBODY® or polypeptide of the disclosure. Yet anothermodification may comprise the introduction of one or more detectablelabels or other signal-generating groups or moieties, depending on theintended use of the labeled NANOBODY®. Suitable labels and techniquesfor attaching, using and detecting them will be clear to the skilledperson and, for example, include, but are not limited to, fluorescentlabels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine andfluorescent metals such as Eu or others metals from the lanthanideseries), phosphorescent labels, chemiluminescent labels orbioluminescent labels (such as luminal, isoluminol, theromaticacridinium ester, imidazole, acridinium salts, oxalate ester, dioxetaneor GFP and its analogs), radio-isotopes, metals, metal chelates ormetallic cations or other metals or metallic cations that areparticularly suited for use in in vivo, in vitro or in situ diagnosisand imaging, as well as chromophores and enzymes (such as malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholine esterase). Other suitable labels will beclear to the skilled person and, for example, include moieties that canbe detected using NMR or ESR spectroscopy. Such labeled NANOBODIES® andpolypeptides of the disclosure may, for example, be used for in vitro,in vivo or in situ assays (including immunoassays known per se, such asELISA, RIA, EIA and other “sandwich assays,” etc.) as well as in vivodiagnostic and imaging purposes, depending on the choice of the specificlabel.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example, to chelate one ofthe metals or metallic cations referred to above. Suitable chelatinggroups include, for example, without limitation,diethyl-enetriaminepentaacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional group may be usedto link the NANOBODY® of the invention to another protein, polypeptideor chemical compound that is bound to the other half of the bindingpair, i.e., through formation of the binding pair. For example, aNANOBODY® hereof may be conjugated to biotin, and linked to anotherprotein, polypeptide, compound or carrier conjugated to avidin orstreptavidin. For example, such a conjugated NANOBODY® may be used as areporter, for example, in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may, for example, also be used to bind the NANOBODY® ofthe invention to a carrier, including carriers suitable forpharmaceutical purposes. One non-limiting example is the liposomalformulations described by Cao and Suresh, Journal of Drug Targeting 8,4:257 (2000). Such binding pairs may also be used to link atherapeutically active agent to the NANOBODY® hereof.

It is expected that the NANOBODIES® and polypeptides of the disclosurewill generally bind to all naturally occurring or synthetic analogs,variants, mutants, alleles, parts and fragments of BACE1, or at least tothose analogs, variants, mutants, alleles, parts and fragments of BACE1,that contain one or more antigenic determinants or epitopes that areessentially the same as the antigenic determinant(s) or epitope(s) towhich the NANOBODIES® and polypeptides of the disclosure bind in BACE1(e.g., in wild-type BACE1). Again, in such a case, the NANOBODIES® andpolypeptides of the disclosure may bind to such analogs, variants,mutants, alleles, parts and fragments with an affinity and/orspecificity that are the same as, or different from (i.e., higher thanor lower than), the affinity and specificity with which the NANOBODIES®hereof bind to (wild-type) BACE1.

It is also included within the scope hereof that the NANOBODIES® andpolypeptides of the disclosure bind to some analogs, variants, mutants,alleles, parts and fragments of BACE1, but not to others. Also, indetermining the degree of sequence identity between two amino acidsequences, the skilled person may take into account so-called“conservative” amino acid substitutions, which can generally bedescribed as amino acid substitutions in which an amino acid residue isreplaced with another amino acid residue of similar chemical structureand which has little or essentially no influence on the function,activity or other biological properties of the polypeptide. Suchconservative amino acid substitutions are well known in the art, forexample, from WO04037999, WO9849185, WO0046383 and WO0109300; and(preferred) types and/or combinations of such substitutions may beselected on the basis of the pertinent teachings from WO04037999, aswell as WO9849185. Such conservative substitutions preferably aresubstitutions in which one amino acid within the following groups(a)-(e) is substituted by another amino acid residue within the samegroup: (a) small aliphatic, nonpolar or slightly polar residues: Ala,Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their(uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively chargedresidues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met,Leu, His, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.Particularly preferred conservative substitutions are as follows: Alainto Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp intoGlu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro;His into Asn or into GIn; His into Leu or into Val; Leu into His or intoVal; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or intoHis; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trpinto Tyr; Tyr into Trp; and/or Phe into Val, into His or into Leu.

Polypeptide therapeutics and, in particular, antibody-basedtherapeutics, have significant potential as drugs because they haveexquisite specificity to their target and a low inherent toxicity.However, it is known by the skilled person that an antibody that hasbeen obtained for a therapeutically useful target requires additionalmodification in order to prepare it for human therapy, so as to avoid anunwanted immunological reaction in a human individual uponadministration. The modification process is commonly termed“humanization.” It is known by the skilled artisan that antibodiesraised in species, other than in humans, require humanization to renderthe antibody therapeutically useful in humans ((1) CDR grafting: ProteinDesign Labs: U.S. Pat. No. 6,180,370, U.S. Pat. No. 5,693,761; GenentechU.S. Pat. No. 6,054,297; Celltech: EP626390, U.S. Pat. No. 5,859,205;(2) Veneering: Xoma: U.S. Pat. No. 5,869,619, U.S. Pat. No. 5,766,886,U.S. Pat. No. 5,821,123). There is a need for a method for producingantibodies that avoids the requirement for substantial humanization orthat completely obviates the need for humanization.

There is a need for a new class of antibodies that have definedframework regions or amino acid residues and that can be administered toa human subject without the requirement for substantial humanization, orthe need for humanization at all. According to one aspect of theinvention, NANOBODIES® are polypeptides that are derived from heavychain antibodies and whose framework regions and complementarydetermining regions are part of a single domain polypeptide. Examples ofsuch heavy chain antibodies include, but are not limited to, naturallyoccurring immunoglobulins devoid of light chains. Such immunoglobulinsare disclosed in WO9404678, for example. The antigen-binding site ofthis unusual class of heavy chain antibodies has a unique structure thatcomprises a single variable domain. For clarity reasons, the variabledomain derived from a heavy chain antibody naturally devoid of lightchain is known herein as a V_(H)H or V_(H)H domain or NANOBODY®. Such aV_(H)H domain peptide can be derived from antibodies raised in Camelidaespecies, for example, in camel, dromedary, llama, alpaca and guanaco.Other species besides Camelidae (e.g., shark, pufferfish) may producefunctional antigen-binding heavy chain antibodies naturally devoid oflight chain. V_(H)H domains derived from such heavy chain antibodies arewithin the scope of the invention.

Camelidae antibodies express a unique, extensive repertoire offunctional heavy chain antibodies that lack light chains. The V_(H)Hmolecules derived from Camelidae antibodies are the smallest intactantigen-binding domains known (approximately 15 kDa, or ten timessmaller than conventional IgG) and, hence, are well suited towarddelivery to dense tissues and for accessing the limited space betweenmacromolecules. Other examples of NANOBODIES® include NANOBODIES®derived from VH domains of conventional four-chain antibodies that havebeen modified by substituting one or more amino acid residues withCamelidae-specific residues (the so-called camelization of heavy chainantibodies, WO9404678). Such positions may preferentially occur at theVH-VL interface and at the so-called Camelidae hallmark residues(WO9404678), comprising positions 37, 44, 45, 47, 103 and 108.NANOBODIES® correspond to small, robust and efficient recognition unitsformed by a single immunoglobulin (Ig) domain.

A “fragment of a NANOBODY®” as used herein refers to less than 100% ofthe sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%etc.), but comprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25 or more amino acids. A fragment is preferably ofsufficient length such that the interaction of interest is maintainedwith affinity of 1×10⁶ M or better. A “fragment” as used herein alsorefers to optional insertions, deletions and substitutions of one ormore amino acids that do not substantially alter the ability of thetarget to bind to a NANOBODY® raised against the wild-type target. Thenumber of amino acid insertions, deletions or substitutions ispreferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70amino acids. One embodiment of the present invention relates to apolypeptide comprising at least one NANOBODY® wherein one or more aminoacid residues have been substituted without substantially altering theantigen binding capacity.

In a particular embodiment, the antibody of the invention is bivalentand formed by bonding together, chemically or by recombinant DNAtechniques, two monovalent single domains of heavy chains. In anotherparticular embodiment, the antibody of the invention is bi-specific andformed by bonding together two variable domains of heavy chains, eachwith a different specificity (i.e., one with a specificity for BACE1 andthe other one with a specificity for a neuron, such as, for example,ICAM5 or telencephalin). Similarly, polypeptides comprising multivalentor multi-specific single domain antibodies are included here asnon-limiting examples.

In yet another embodiment, a single domain antibody that is capable ofpreventing the uptake of BACE1 can be used as a medicament. In yetanother embodiment, a single domain antibody that comprises at least oneof the complementarity-determining regions (CDRs) with an amino acidsequence selected from the group comprising SEQ ID NOS:29-37 can be usedas a medicament. In yet another embodiment, a single domain antibody,which amino acid comprises SEQ ID NOS:6, 22 or 26, can be used as amedicament.

In yet another embodiment, a single domain antibody that is capable ofpreventing the uptake of pro-BACE1 can be used for the manufacture of amedicament to treat diseases associated with an overexpression of BACE1.An example of a disease where an overexpression of BACE1 occurs isAlzheimer's disease. In general, “therapeutically effective amount,”“therapeutically effective dose” and “effective amount” means the amountneeded to achieve the desired result or results (inhibiting BACE1binding; treating or preventing Alzheimer's disease). One of ordinaryskill in the art will recognize that the potency and, therefore, an“effective amount” can vary for the NANOBODY® that inhibits BACE1binding used in the invention. One skilled in the art can readily assessthe potency of the NANOBODY®. By “pharmaceutically acceptable” is meanta material that is not biologically or otherwise undesirable, i.e., thematerial may be administered to an individual along with the compoundwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

The term “medicament to treat” relates to a composition comprisingantibodies as described above and a pharmaceutically acceptable carrieror excipient (both terms can be used interchangeably) to treat or toprevent diseases as described herein. The administration of a NANOBODY®as described above or a pharmaceutically acceptable salt thereof may beby way of oral, inhaled or parenteral administration. In particularembodiments, the NANOBODY® is delivered through intrathecal orintracerebroventricular administration. The active compound may beadministered alone or preferably formulated as a pharmaceuticalcomposition.

An amount effective to treat Alzheimer's disease that expresses theantigen recognized by the NANOBODY® depends on the usual factors, suchas the nature and severity of the disorder being treated and the weightof the mammal. However, a unit dose will normally be in the range of0.01 to 50 mg, for example, 0.01 to 10 mg, or 0.05 to 2 mg of NANOBODY®or a pharmaceutically acceptable salt thereof. Unit doses will normallybe administered once or more than once a day, for example, two, three,or four times a day, more usually one to three times a day, such thatthe total daily dose is normally in the range of 0.0001 to 1 mg/kg; thusa suitable total daily dose for a 70 kg adult is 0.01 to 50 mg, forexample, 0.01 to 10 mg or more, usually 0.05 to 10 mg.

In certain embodiments, the compound or a pharmaceutically acceptablesalt thereof is administered in the form of a unit-dose composition,such as a unit dose oral, parenteral, or inhaled composition. Suchcompositions are prepared by admixture and are suitably adapted fororal, inhaled or parenteral administration and, as such, may be in theform of tablets, capsules, oral liquid preparations, powders, granules,lozenges, reconstitutable powders, injectable and infusable solutions orsuspensions or suppositories or aerosols. Tablets and capsules for oraladministration are usually presented in a unit dose, and containconventional excipients such as binding agents, fillers, diluents,tableting agents, lubricants, disintegrants, colorants, flavorings, andwetting agents. The tablets may be coated according to well-knownmethods in the art. Suitable fillers for use include cellulose,mannitol, lactose and other similar agents. Suitable disintegrantsinclude starch, polyvinylpyrrolidone and starch derivatives such assodium starch glycolate. Suitable lubricants include, for example,magnesium stearate. Suitable pharmaceutically acceptable wetting agentsinclude sodium lauryl sulphate. These solid oral compositions may beprepared by conventional methods of blending, filling, tableting, or thelike. Repeated blending operations may be used to distribute the activeagent throughout those compositions employing large quantities offillers. Such operations are, of course, conventional in the art.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups, or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example, sorbitol,syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel or hydrogenated edible fats,emulsifying agents, for example, lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample, almond oil, fractionated coconut oil, oily esters such asesters of glycerine, propylene glycol, or ethyl alcohol; preservatives,for example, methyl or propyl p-hydroxybenzoate or sorbic acid, and, ifdesired, conventional flavoring or coloring agents. Oral formulationsalso include conventional sustained release formulations, such astablets or granules having an enteric coating.

Preferably, compositions for inhalation are presented for administrationto the respiratory tract as a snuff or an aerosol or solution for anebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of active compound suitably have diameters of less than 50microns, preferably less than 10 microns, for example, between 1 and 5microns, such as between 2 and 5 microns. A favored inhaled dose will bein the range of 0.05 to 2 mg, for example, 0.05 to 0.5 mg, 0.1 to 1 mgor 0.5 to 2 mg.

For parenteral administration, fluid unit dose forms are preparedcontaining a compound of the present invention and a sterile vehicle.The active compound, depending on the vehicle and the concentration, canbe either suspended or dissolved. Parenteral solutions are normallyprepared by dissolving the compound in a vehicle and filter sterilizingbefore filling into a suitable vial or ampoule and sealing.Advantageously, adjuvants such as a local anesthetic, preservatives andbuffering agents are also dissolved in the vehicle. To enhance thestability, the composition can be frozen after filling into the vial andthe water removed under vacuum. Parenteral suspensions are prepared insubstantially the same manner except that the compound is suspended inthe vehicle instead of being dissolved and sterilized by exposure toethylene oxide before suspending in the sterile vehicle.

Advantageously, a surfactant or wetting agent is included in thecomposition to facilitate uniform distribution of the active compound.Where appropriate, small amounts of bronchodilators, for example,sympathomimetic amines such as isoprenaline, isoetharine, salbutamol,phenylephrine and ephedrine; xanthine derivatives such as theophyllineand aminophylline; corticosteroids such as prednisolone; and adrenalstimulants such as ACTH, may be included. As is common practice, thecompositions will usually be accompanied by written or printeddirections for use in the medical treatment concerned.

In yet another embodiment, one or more single domain antibodies of theinvention can be linked (optionally, via one or more suitable linkersequences) to one or more (such as two and preferably one) amino acidsequences that allow the resulting polypeptide of the invention to crossthe blood brain barrier. In particular, the one or more amino acidsequences that allow the resulting polypeptides of the invention tocross the blood brain barrier may be one or more (such as two andpreferably one) NANOBODIES®, such as the NANOBODIES® described in WO02/057445, of which FC44 (SEQ ID NO:189 of WO 06/040153) and FC5 (SEQ IDNO:190 of WO 06/040154) are preferred examples.

The present invention further provides a pharmaceutical composition foruse in the treatment and/or prophylaxis of herein-described disorders,which comprises a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable solvate thereof, and, if required, apharmaceutically acceptable carrier thereof.

It should be clear that the therapeutic method hereof for addressingAlzheimer's disease can also be used in combination with any other ADdisease therapy known in the art, such as gamma-secretase inhibitors orother beta-secretase inhibitors.

In a particular embodiment, the single domain antibodies hereof can beused for the preparation of a diagnostic assay. BACE1 can be detected ina variety of cells and tissues, especially in brain cells and tissues,wherein the degree of expression corroborates with the severity ofAlzheimer's disease. Therefore, there is provided a method of in situdetecting localization and distribution of BACE1 expression in abiological sample. The method comprises the step of reacting thebiological sample with a detectable BACE1 NANOBODY® and detecting thelocalization and distribution of the detectable NANOBODY®. The term“biological sample” refers to cells and tissues, including, but notlimited to, brain cells and tissues. The term further relates to bodyfluids.

Therefore, there is provided a method of detecting BACE1 protein in abody fluid of a patient. The method comprises the steps of reacting thebody fluid with an anti-BACE1 NANOBODY® hereof and monitoring thereaction. The body fluid is, for example, plasma, urine, cerebrospinalfluid, pleural effusions or saliva. Monitoring the reaction may beeffected by having the NANOBODY® labeled with a detectable moiety, or touse its constant region as an inherent detectable moiety, to which asecond antibody, which includes a detectable moiety, can specificallybind. CSF BACE1 can, for example, be detected in patients suffering fromAlzheimer's disease. According to a preferred embodiment of the presentinvention, reacting the body fluid with the anti-BACE1 NANOBODY® iseffected in solution.

Alternatively, reacting the body fluid with the anti-BACE1 NANOBODY® iseffected on a substrate capable of adsorbing proteins present in thebody fluid, all as well known in the art of antibody-based diagnosis.Further, according to the disclosure, there is provided a method ofdetecting the presence, absence or level of BACE1 protein in abiological sample. The method comprises the following steps. First,proteins are extracted from the biological sample, thereby a pluralityof proteins are obtained. The protein extract may be a crude extract andcan also include non-proteinaceous material. Second, the proteins aresize separated, e.g., by electrophoresis, gel filtration, etc. Fourth,the size-separated proteins are interacted with an anti-BACE1 NANOBODY®.Finally, the presence, absence or level of the interacted anti-BACE1NANOBODY® is detected. In case of gel electrophoresis, the interactionwith the NANOBODY® is typically performed following blotting of thesize-separated proteins onto a solid support (membrane).

The following examples more fully illustrate the disclosure. Startingmaterials and reagents disclosed below are known to those skilled in theart, and are available commercially or can be prepared using well-knowntechniques.

EXAMPLES 1. Generation and Isolation of BACE1-Specific NANOBODIES®

To generate BACE1-specific antibodies, a dromedary was immunized sixtimes with recombinant human BACE1 over a period of about six weeks.After this period, a BACE1-specific humoral response, as assessed byELISA, was observed for each of the three different IgG subclasses thatexist in Camelidae, namely the conventional IgG1 molecules and the heavychain-only subclasses IgG2 and IgG3 (IgG classes reviewed in Conrath etal., 2003). The variable chain of the HCAbs (V_(H)H), which contains theantigen-binding fragment, was amplified from isolated dromedarylymphocytes and cloned into a pHEN4 phagemid vector to generate alibrary of 4×10⁷ individual transformants. After rescuing this bank withM13K07 helper phages, the V_(H)H repertoire was expressed on the surfaceof bacteriophages. With these phages, BACE1-specific V_(H)Hs could beisolated from the whole V_(H)H pool using panning, an in vitro selectiontechnique (reviewed in Smith and Petrenko, 1997). For this, the phageswere incubated onto a solid phase passively coated with the immunogen.After washing, bound phages were eluted and used to infect exponentiallygrowing E. coli TG1 cells to produce new virions. These virions wereused in a next selection round in order to enrich for BACE1-specificbinders. After two to three consecutive rounds of panning, individualcolonies were randomly picked and incubated with IPTG to induceexpression of the NANOBODIES®. The V_(H)H protein fragments wereextracted from the bacterial periplasm and tested individually by ELISAfor their ability to interact with BACE1. The positive scoring cloneswere sequenced and, as such, twenty different specific NANOBODIES® wereidentified.

2. Sequence Analysis of the BACE1-Binders

Fourteen out of the twenty selected BACE1-binders are clearly derivedfrom V_(H)H germ-line genes: Nb_B1, Nb_B2, Nb_B3, Nb_B5, Nb_B8, Nb_B9,Nb_B10, Nb_B11, Nb_B12, Nb_B15, Nb_B16, Nb_B21, Nb_B25, and Nb_B26,corresponding with SEQ ID NOS:1-14, respectively (FIG. 1). The aminoacid sequence of their framework-2 (FR2) region resembles that of atypical V_(H)H FR2 (Muyldermans et al., 1994), with residues F/Y, E/Q,R/C and G at positions 37, 44, 45 and 47, respectively (numberingaccording to Kabat et al., 1991). However, the remaining six antibodyfragments, Nb_B4, Nb_B6, Nb_B7, Nb_B13, Nb_B14 and Nb_B24 (correspondingwith SEQ ID NOS:38-43, respectively), seem to originate fromconventional antibody germ-line genes, since they contain theV₃₇G₄₄L₄₅W₄₇ tetrad, a typical hallmark that distinguishes the variabledomain of the heavy chain of conventional antibodies (VH) from V_(H)Hfragments at the germline level. These hallmark residues are criticallyrequired in H₂-L₂ antibodies for the association of the heavy chain witha light chain.

Due to the high sequence similarity of the six V_(H)-like NANOBODIES®,it is most likely they are all derived from one and the same B celllineage. The differences in amino acid sequence could be the result ofthe ongoing somatic hypermutation of the antibody gene fragments inmaturing B cells and the subsequent antigen-driven selection, acontinuous process leading to ever better fitting antibodies. The sixV_(H)-like NANOBODIES® also differ from the other binders in that theycontain a leucine residue at position 11 in their framework-1 (FR1),another characteristic of V_(H) genes that is important for theinteraction with a light chain (Lesk and Chothia, 1988; Padlan, 1994).

In a typical V_(H)H FR1, this Leu residue is often replaced by a smallerand hydrophilic residue, usually a serine, as seen in the 14 BACE1binders with a true V_(H)H motif. The V_(H)-like molecules all have ashort CDR3 of only six amino acids, whereas, the other binders havesignificantly larger H3 loops, ranging from 13 to 21 residues, with anaverage length of 17. This is consistent with the average V_(H)H-CDR3length of 15 to 16 residues reported before in literature (reviewed inMuyldermans and Lauwereys, 1999).

In general, the CDR2 and CDR1 of V_(H)Hs consist of 16 to 17 and 10residues, respectively, but about 30% of dromedary V_(H)H cDNAs werereported to be off-sized. This does not adversely affect their function,but instead, even increases the antigen-binding repertoire (Nguyen etal., 2000). Unusual CDR1 and CDR2 lengths are also observed for ourBACE1 binders. The CDR2 of Nb_B15 contains 19 residues due to a tandemrepetition of two amino acids, whereas, that of Nb_B21 consists of 18residues. Aberrant CDR1 sizes are found in Nb_B1, Nb_B15 and Nb_B16 dueto an insertion, a deletion of one amino acid and a deletion of tworesidues, respectively. Finally, Nb_B25 has an unusually longframework-3 region with a tandem repetition of two amino acid residues.Deviating lengths in the BACE1-binders are due to changes located atthree typical V_(H)H insertion/deletion hot spots, surrounding residues30±3, 54±3 and 74±1. These hot spots can be found within or at theborder of peculiar DNA sequences, such as palindromic sequences(corresponding to residues 30-33 and 54-57) or heptamer-like sequencesof an Ig recombination signal (often found at residues 76-78) (Nguyen etal., 2000).

Besides the conserved disulphide bridge between Cys₂₂ and Cys₉₂, extranon-canonical cysteine residues do not frequently occur in conventionalantibodies, although they are not totally excluded. However, anadditional pair of cysteines is encountered in 75% of reported dromedaryV_(H)Hs (Arbabi Ghahroudi et al., 1997; Lauwereys et al., 1998; Conrathet al., 2001a; Saerens et al., 2004). One of these extra cysteineresidues is typically located within the CDR3 loop, whereas, the otherone can be found either on position 30, 32 or 33 within the CDR1 or atposition 45 in FR2. Since the V_(H)H CDR3 loop folds back onto theCDR1-FR2 region, the two cysteine residues come into contact distanceand are likely engaged into an interloop disulphide bond thatcross-links the antigen-binding loops (Desmyter et al., 1996). Such abond reduces flexibility of the long CDR3 loop and thus providesincreased stability. Besides, the interloop bond might lead to aconstrained, but new conformation of the CDR loops, thereby increasingthe antigen-binding repertoire.

Compared to the percentages known from literature, there is a lowincidence of additional cysteines in the BACE1 binders. A putativeadditional disulphide bond is only present in four out of the 14NANOBODIES® with V_(H)H motif. Nb_B25 has a cysteine residue at position33 within the CDR1; Nb_B9 has one at position 32; and in Nb_B5, anadditional bridge will probably be formed between Cys₄₅ and the CDR3. Acysteine at position 53, as seen in Nb_B12, has been described so farfor neither dromedary, nor llama V_(H)Hs.

3. Defining Affinities of the BACE1-Binders for their Immunogen at pH7.0 and pH 5.0

The cDNAs of the 20 isolated BACE1-binders were subcloned into the pHEN6prokaryotic expression vector and expressed in E. coli WK6 cells toproduce his-tagged soluble proteins. The recombinant V_(H)Hs weresubsequently purified by Ni-NTA affinity chromatography, followed bysize-exclusion chromatography. The expression levels of the distinctclones varied between 1 and 15 mg per liter of culture medium. Theaffinity of all V_(H)Hs for BACE1 was determined quantitatively usingthe surface plasmon resonance technology on Biacore 3000. Each of thedifferent V_(H)Hs was injected at concentrations ranging from 0 to 0.5μM on a chip onto which BACE1 was coupled. Binding was evaluated both atpH 7.0 and pH 5.0. Measurements at pH 5.0 were included because a firminteraction between BACE1 and a V_(H)H should be preserved at this pH,since the endosomal compartment with its slightly acidic content wasreported to be the major subcellular site of β-site cleavage of APP(Koo, 1994). Besides, BACE1 was shown to have optimal β-secretaseactivity at about pH 5.0 in vitro (Sinha et al., 1999; Vassar et al.,1999; Yan et al., 1999; Lin et al., 2000). The dissociation constantsobtained for all V_(H)Hs vary between 4 and 669 nM at pH 7.0 and between4.2 nM and 6.8 μM at pH 5.0 (FIG. 2, Panel A). The majority of thebinders have dissociation constants between 10 and 100 nM at both pHconditions.

4. Capacity of the Different V_(H)Hs to Pull Down Native BACE1

For the immunization of the dromedary, the isolation of theBACE1-specific binders during panning and the in vitro affinitymeasurements by Biacore, we used recombinant human soluble BACE1,completely devoid of carbohydrate chains. This recombinant protein,supplied by Dr. S. Masure (Johnson & Johnson, Beerse, Belgium) wasobtained from an insect cell expression system using slightly truncatedBACE1 cDNA in which the four N-glycosylation sites and the wholemembrane anchor were removed (Bruinzeel et al., 2002). It is notunthinkable that epitopes that are easily accessible in the “naked”BACE1, used for the immunization, are shielded by glycan chains or otherpost-translational modifications of BACE1 proteins generated inmammalian cells. Therefore, we wondered whether the selected binderswould all be able to recognize glycosylated BACE1 expressed in mammaliancells.

To test this, 2 μg of his-tagged V_(H)H molecules were incubated with 4μg of total protein extract from COS cells transiently transfected withhuman BACE1 cDNA. Nickel-beads were subsequently used to pull down theV_(H)H molecules together with the bound proteins. After extensivewashing, bound proteins were eluted, separated by SDS-PAGE and BACE1protein was detected by Western blotting using a rabbit polyclonalBACE1-specific antibody (ProSci, 2253) (FIG. 2, Panel B). V_(H)Hproteins raised against either Aβ (Nb_Aβ3) or beta-lactamase BCII 569/H(Nb_BCIILP) (Conrath et al., 2001a), were used as negative controls andwere unable to capture BACE1 from the cell lysate, as expected. Fivebinders, Nb_B7, Nb_B9, Nb_B10, Nb_B13 and Nb_B24 have the highestefficacy to pull down BACE1 compared to the other NANOBODIES®. ForNb_B3, Nb_B8, Nb_B12 and Nb_B21 at the best a trifling trace ofcoprecipitated BACE1 can be detected after overnight exposure.

Note that in the group of the 6 V_(H)-like NANOBODIES® (Nb_B4, Nb_B6,Nb_B7, Nb_B13, Nb_B14 and Nb_B24), huge differences are observed in theability of each NANOBODY® to bind to glycosylated BACE1, even thoughthey probably originated from the same B-cell lineage. Despite a highoverall sequence similarity of about 90%, Nb_B13 and Nb_B14 share only70% of amino acids in their antigen-binding CDR regions and thisdifference apparently is sufficient to adversely affect the affinity ofNb_B14 for its antigen when compared to Nb_B13.

5. Effect of Ectopic Expression of the NANOBODIES® on BACE Inhibition

In a next step, we decided to express some of the V_(H)Hs into mammaliancells. Thereto, COST-B1 cells, stably expressing low levels of BACE1,were co-transfected with APPSw and either Nb_B26 or Nb_B9. Control cellswere either transfected with empty vector or with APPSw alone. The cDNAs(of Nb_B26 or Nb_B9) were cloned into a eukaryotic expression vector,downstream of the signal peptide of BACE1 and with a myc-epitope tag atits C-terminus (FIG. 3, Panel A). The signal sequence ensurestranslocation of the newly formed protein into the secretory pathway,where the V_(H)H should encounter its antigen, the ectodomain of BACE1.APP_(Sw) and the two V_(H)Hs were co-transfected into COS cells stablyexpressing low levels of human BACE1 (COS-B1 cells). These cells havedetectable, but not saturated, levels of β-secretase activity and areeasily transfected using liposome-based transfection reagents. Two daysafter transfection, cell extracts were prepared, proteins were separatedby SDS-PAGE and transferred to a nitrocellulose membrane. Using rabbitpolyclonal B63.1 as a primary antibody and GARIR, an infrared-coupledsecondary antibody, the APP C-terminal fragments were visualized andquantified by the ODYSSEY® Infrared Imaging System (FIG. 3, Panels B andC). Again, Nb_B26 had no effect on APP processing. The ratio of β-CTF ontotal APP CTFs is equal to that of non-treated cells (FIG. 3, Panel C).Nb_B9 consistently decreased β-secretase activity by about 30%(p<0.001), even though it was expressed at much lower levels than Nb_B26(FIG. 3, Panel B). This decrease occurred in the absence of any effecton BACE1 protein levels, ruling out the possibility that the NANOBODY®affects BACE1 protein stability.

6. Effect of Addition of Extracellular NANOBODIES® on APP Processing inCells

In a next step, NANOBODY® Nb_B9 was tested as to whether it could alsoaffect APP processing when added to culture medium of cells. At leastpart of BACE1 is directed to the plasma membrane before being targetedto endosomes, so BACE1-specific antibodies could potentially bind to theectodomain at the cell surface and be smuggled inside cells byco-internalization with their antigen. Since β-secretase cleavage ofwild-type APP predominantly occurs within the endosomal compartments,neutralizing the enzyme's activity from the plasma membrane might besufficient to decrease β-site APP proteolysis. SH-SY5Y cells,neuroblastoma cells with relatively high endogenous BACE1 activity, wereinfected with recombinant adenoviruses containing the cDNA encodingeither human APP wild-type or the FAD APP_(Sw) mutant. The FAD mutantwas included since it is a much better BACE1 substrate, which enableseasier detection of Aβ and β-CTF. However, the majority of APP_(Sw) hasbeen shown to be cleaved at the β-site in the secretory pathway beforereaching the plasma membrane (Martin et al., 1995; Thinakaran et al.,1996), so BACE1-neutralizing V_(H)Hs binding at the cell surface mightnot be capable of preventing β-site APP_(Sw) cleavage.

Two days after adenoviral infection, the SH-SY5Y cells wereradioactively labeled and incubated with 2 μM Nb_B9 for six hours. Theconditioned medium was used to immunoprecipitate secreted Aβ, whereas,APP full-length and C-terminal fragments were pulled down from celllysates. APP fragments were separated by SDS-PAGE. Gels were fixed,dried and analyzed by phosphorimaging. The presence of Nb_B9 causedclear detectable change in amounts of β-CTF or Aβ compared tonon-treated cells (FIG. 4).

7. Isolation of Other BACE1-Specific NANOBODIES®

Further, a new screening of the V_(H)H phage libraries was performedusing a different panning strategy, while Nb_B9 was included for furtheranalysis. Phage pannings of the two V_(H)H libraries were performedusing biotinylated antigen (the ectodomain of human BACE1). After threerounds of consecutive panning, 500 single colonies were randomly pickedfor phage ELISA screening. One hundred fifty-eight out of 500 coloniesscored positive in phage ELISA screening. The positive colonies werefurther screened by periplasmic extract ELISA; 44 colonies out the 158colonies were scored positive. The positive colonies isolated fromperiplasmic extract ELISA screening were analyzed by PCR and restrictionenzyme digestion to group them according to restriction pattern and forfurther sequencing analysis. Fourteen new V_(H)Hs were identified fromthe screening.

The alignment of the V_(H)Hs sequence was listed in FIG. 5. Among theseV_(H)Hs, ten clones (1B3, 10C2, 12B6, 10B5, 13A5, 2C6, 6A4, 10C4, 13B6and 1A4 (SEQ ID NOS:15-24, respectively)) were isolated from thedromedary libraries, and four clones (2B6, 4A2, 1D4 and 9D3 (SEQ IDNOS:25-28, respectively)) were isolated from the llama library. The cDNAof these clones were subcloned into expression vector pHEN6 and V_(H)Hantibodies were then purified for functional assay tests.

8. BACE1-Specific NANOBODIES® Inhibit BACE1 Activity in a Cellular Assayand Modulate BACE1 Activity in a Cell-Free Enzymatic Assay

All 15 V_(H)Hs (14 new V_(H)Hs+Nb_B9) were first tested in a cellularassay by adding them to the medium of SH-SY5Y cells stably expressingAPPwt at a final concentration of 20 μM. As shown in FIG. 6, cellstreated with V_(H)Hs B9, 10C4, 4A2 for 24 hours were shown to decreasesAPPIβ generation while sAPPα levels in the conditioned medium remainedthe same as that of control, suggesting BACE1 activity was inhibited bythese V_(H)Hs in the cellular assay.

In parallel, the capacity of the V_(H)Hs to modulate β-secretaseactivity was tested in an in vitro β-secretase assay that is based onthe Fluorescence Resonance Energy Transfer (FRET) technology. This assaymakes use of a synthetic peptide substrate that mimics the BACE1cleavage site of APP and is coupled to a fluorophore on its N-terminusand a fluorescence acceptor on its C-terminus. The light emitted by thefluorophore is absorbed by the fluorescence acceptor as long as thesetwo moieties are in close proximity. Only upon proteolysis, whenrecombinant BACE1 is added to the synthetic substrate, energy transferno longer occurs and the amount of light emitted, which is linearlyrelated to the amount of cleaved product and, hence, to the β-secretaseactivity, can be measured. All V_(H)Hs were tested by this BACE1 FRETassay at a final concentration of 10 μM.

As shown in FIG. 7, 10C4 and 4A2, the two candidate BACE1 inhibitorsidentified in the cellular assays, inhibited BACE1 activity in the FRETassay. Interestingly, B9, the candidate inhibitor isolated from thecellular assay, was shown to increase 260% of BACE1 activity in the FRETassay. Another clone, 1B3 was also shown to increase 125% of BACE1activity in the FRET assay, although it had no apparent effect on BACE1in the cellular assay. The remaining V_(H)Hs had no or negligibleeffects on BACE1 activity in the FRET assay.

The dose-response curves of 10C4, 4A2 and B9 on BACE1 activity wereestablished by FRET assay. As shown in FIG. 8, 10C4 could inhibitmaximal ˜70% of BACE1 activity and the IC50 was 150 nM. 4A2 couldinhibit maximal ˜40% of BACE1 activity and the IC50 was 1.2 μM. B9 couldincrease BACE1 activity up to 3.5 times with ˜100 nM concentration, andthe EC50 in the response curve was 4.1 nM.

The contradictory results from B9 modulating BACE1 activity in oppositeways in the cellular assay and the FRET assay implicates that B9 mighthave different effects on BACE1 cleavage of a large substrate or a smallsubstrate (APP as the cellular substrate for BACE1 contains 695 aminoacids while the peptide substrate in FRET assay contains only ten aminoacids). Therefore, it was tested whether B9 could inhibit BACE1 cleavageof a big substrate in another cell-free enzymatic assay MBP-ELISA, whichuses the maltose binding protein connected to the C-terminal 125 aminoacids of APPswe (MBP-APPswe-C125) as BACE1 substrate. As shown in FIG.9, B9 inhibited BACE1 cleavage of MBP-APPswe-C125 in a dose-dependentmanner, and could inhibit up to 95% of BACE1 activity. The results ofthis assay indicate that B9 is an inhibitor of BACE1 when using a bigpeptide substrate. So, V_(H)H Nb_B9, instead of being an active sitebinder, was more likely a steric inhibitor for BACE1. V_(H)H Nb_B9 couldbind to an allosteric site on BACE1, thus stimulating BACE1 cleavage ofsmall substrates that can still reach the cleavage site, but blockingaccess of big substrates, like APP to BACE1 by steric hindrance.

9. Affinity Analysis of the BACE1-Specific NANOBODIES®

The binding affinities of V_(H)Hs B9, 10C4 and 4A2 to human BACE1ectodomain were analyzed by Biacore. As shown in Table 2 (left), B9 hadthe best affinity among the three inhibitory V_(H)Hs, with a Kd of 3.67nM at pH 7.0. 10C4 and 4A2 had affinities of 74.7 nM and 48.2 nM,respectively, which are all within the normal range of affinities forV_(H)H antibodies.

Further, it was tested if the affinities of the V_(H)Hs were stable atpH 4.5, at which BACE1 has its optimal activity. As shown in Table 2(right), there was no significant change in the affinities of the threeV_(H)Hs at pH 4.5 compared to that in neutral pH, indicating that allthree V_(H)Hs have binding affinities to BACE1 that were acidic stable.

TABLE 2 V_(H)H affinities to human BACE1 at pH 7.0 and pH 4.5 pH 7.0 pH4.5 k_(on) k_(off) K_(D) k_(on) k_(off) K_(D) (M⁻¹s⁻¹) (s⁻¹) (nM)(M⁻¹s⁻¹) (s⁻¹) (nM) Nb_B9 2.67E+05 9.80E−04 3.67 6.62E+05 1.30E−03 1.96Nb_4A2 4.79E+05 2.31E−02 48.2 3.97E+05 8.41E−03 21.2 Nb_10C4 1.06E+057.92E−03 74.7 4.51E+05 1.25E−02 27.7

The cross-reactivity of three V_(H)Hs to mouse BACE1 was investigated inanticipation of tests in primary cultures of mouse neurons. As shown inTable 3, both at neutral pH and acidic pH condition, all three V_(H)Hscross-reacted with mouse BACE1, and their affinities to mouse BACE1 werewithin the same range of affinities as those measured with human BACE1.

TABLE 3 V_(H)H affinities to mouse BACE1 at pH 7.0 and pH 4.5 pH 7.0 pH4.5 k_(on) k_(off) K_(D) k_(on) k_(off) K_(D) (M⁻¹s⁻¹) (s⁻¹) (nM)(M⁻¹s⁻¹) (s⁻¹) (nM) Nb_B9 5.02E+05 8.90E−04 1.77 1.06E+06 1.18E−03 1.11Nb_4A2 1.65E+05 5.81E−03 35.2 2.51E+05 2.16E−03 8.61 Nb_10C4 1.90E+057.97E−03 41.9 9.84E+05 9.88E−03 10

10. BACE1-Specific NANOBODIES® Inhibit BACE1 Cleavage of APPwt inPrimary Cultured Mouse Neurons

V_(H)Hs Nb_B9 and Nb_4A2 were tested in the neuronal cell culture assay(FIGS. 10 and 11). Primary cultured neurons from wild-type mice weretransduced with APPwt by Semliki Forest Virus (SFV), and then treatedwith purified V_(H)H Nb_B9 or Nb_4A2 by adding to the medium serialdilutions (V_(H)Hs were first dissolved and diluted in PBS). Neuroncultures were metabolic labeled for six hours. CTFβ, sAPPβ and Aβ werelater analyzed as readout of BACE1 activity.

As shown in FIG. 10, Nb_B9 and Nb_4A2 inhibited BACE1 cleavage of APPreflected in the decrease of A13, sAPPβ, and CTFβ signals, whilefull-length APP and sAPPα levels remained at the same level as that ofthe control. The dose-response curve of Nb_B9 in neuron assay (FIG. 11)was established by quantification of the CTFβ level, which showed Nb_B9inhibited BACE1 activity in a dose-dependent manner, with maximalinhibition effect around 57% BACE1 activity and the IC50 was around 500nM.

11. Validation of BACE1-Inhibitory NANOBODIES® in Mouse Model

Camel single domain antibodies, the minimal-sized antibodies, which havesuperior properties for intracellular expression and function, includingsolubility, stability and functionality without the requirement forassociation between heavy and light chains of conventional antibodies,are candidate therapeutic molecules for in vivo gene delivery. The BACE1inhibitory V_(H)H Nb_B9 is tested in a transgenic mouse model ofAlzheimer's disease through viral vector-mediated gene delivery.Adeno-associated virus (AAV), one of the most effective vehicles forgene delivery to the central nervous system, is used in thisexperimentation. The cDNA of V_(H)H Nb_B9, fused with a signal peptidefrom BACE1 at its N-terminal and a Myc-tag at its C-terminal, wasconstructed into an AAV vector. The AAV vector used here contains ahybrid cytomegalovirus/chicken β-actin promoter and a wood-chuckpost-transcriptional regulatory element, which is an optimized cassettefor driving protein expression in neurons (Björklund et al., 2000).

For in vivo testing, Dutch-mutant APP transgenic mice are used, whichoverexpress E693Q-mutated human APP under the control of aneuron-specific Thyl promoter element (Herzig et al., 2004). The E693QDutch mutation site on human APP is 21 amino acid residues behind BACE1cleavage site, which does not interfere with APP processing by BACE1.Transgenic mice overexpressing the Dutch-mutant APP generatepredominantly Aβ40 peptide, which is used as readout for BACE1 activityfor in vivo test of V_(H)H Nb_B9. Two administrative routes, includingstereotactic injection to the hippocampus region of adult mouse brain(Fukuchi et al., 2006) and intracranial injection to neonatal mousebrain (Levites et al., 2006) are used for the delivery of AAV vectorpackaged V_(H)H Nb_B9. AAV vector packaged GFP and V_(H)H Nb_B24 areused as negative controls.

Materials and Methods Cell Culture

COS, BHK, MEF, CHO, HEK-APP_(Sw), N2A and HeLa cells were cultured at37° C. in a 5% CO₂ environment in Dulbecco's modified Eagle'smedium/nutrient mixture F-12 (1:1) (Gibco) supplemented with 10% (v/v)Fetal Bovine Serum (FBS) (Hyclone). The HEK-APP_(Sw) cells were kindlyprovided by Prof C. Haass (Adolf Butenandt Institute, Ludwig-MaximiliansUniversity, Munich, Germany). For transient liposome-basedtransfections, a mix of FuGENE 6 (Roche Applied Science) and plasmid DNAwith a ratio of 3:1 (in μl and μg, respectively) was added to a culturedish containing a 50% to 80% confluent monolayer of cells, according tothe manufacturer's instructions. COS-hBACE1 stable cells were obtainedafter transient transfection of COS cells with pcDNA3.1zeo-hBACE1 andselection in 400 μg/ml zeocin (Invitrogen). SH-SY5Y cells were grown inDMEM GLuTAMAX® 4500 mg/l D-glucose, 1 mM Sodium pyruvate (Gibco),supplemented with 15% (v/v) FBS.

Primary cortical neuronal cultures were isolated from E14 mouse embryos(according to Goslin and Banker, 1991). Briefly, dissected braincortices were trypsinized with 0.25% trypsin in HBSS medium (Gibco),pelleted and transferred to DMEM (Invitrogen, San Diego, Calif.),supplemented with 10% (v/v) FBS and dissociated by passing them throughPasteur pipettes of decreasing diameters. Dispersed cells were collectedby centrifugation and plated on poly-L-lysine (Sigma)-coated dishes andmaintained in neurobasal medium (Gibco) supplemented with 0.5 μML-glutamine (Invitrogen) and 2% (v/v) B27 Serum-free Supplement (Gibco).Cytosine arabinoside (5 μM) was added 24 hours after plating to preventproliferation of glial cells.

Metabolic Labeling and Immunoprecipitation of APP Fragments

Cells were washed in Met-free or Met/Cys-free medium (GIBCO) andradioactively labeled in the appropriate medium containing,respectively, 100 μCi ³⁵S Met or ³⁵S Met/Cys (Trans ³⁵S Label, MPBiomedicals, Irvine, Calif.). In case of incubations with FK-506,rapamycin and Nb_B26 (2.1 μM), compounds were added to the labelingmedium. After six hours incubation, the culture supernatant wascollected as a source of secreted Aβ or sAPPIβ and centrifuged to removedetached cells. Cells were lysed in DIP buffer (20 mM Tris-HCl pH 7.4,150 mM NaCl, 1% TRITON® X-100, 1% sodium desoxycholate, 0.1% SDS),except for the HEK-APP_(Sw) cells, which were lysed in Tris bufferedsaline (TBS: 150 mM NaCl, 20 mM Tris-HCl, pH 7.5), containing 1% TRITON®X-100 and a cocktail of protease inhibitors (Complete, Roche). Thislysis buffer still allows determination of protein concentration(Bio-Rad Protein Assay) to analyze efficiency of RNA interference onequal amounts of protein extract.

APP full-length and APP C-terminal stubs were precipitated from cellextracts using the APP C-terminal antibodies B63.1, B11/4 or B12/6(1:200) and immunocomplexes were captured by protein G-sepharose. For Aβspecies, samples of the cell-conditioned medium were incubated witheither B7/8 or 4G8 (1:200). For the neurons overexpressing differentBACE1 mutants, BACE1 proteins were precipitated from cell extracts withB45.1.

Immunoprecipitates were washed extensively in DIP buffer, followed byone washing step in TBS 1/3, eluted in LDS sample buffer (Invitrogen)supplemented with 1% β-mercapto ethanol and separated on 10% NuPAGE® gel(Novex) run in MES buffer for the APP fragments and MOPS for the BACE1mutants. Gels were fixed, dried and exposed to a phosphor-imagingscreen. Intensity of radioactive bands was quantified usingPhosphorImaging (Typhoon, PerkinElmer) and the IMAGEQUANT® softwarepackage.

To detect sAPPβ, samples of conditioned medium were subjected toSDS-PAGE and Western blotting using B53/4 antibody.

Deglycosylation Experiments

Cells were harvested in Dulbecco's PBS (GIBCO), pelleted and lysed in100 mM phosphate buffer at pH 5.8 for EndoH treatment (46% of 0.2 MNaH₂PO₄, 4% of 0.2 M Na₂HPO₄ and 50% water) and pH 7.4 for EndoF (9.5%of 0.2 M NaH₂PO₄, 40.5% of 0.2 M Na₂HPO₄ and 50% water), supplementedwith 0.1% SDS, 0.5% TRITON® X-100, 0.5% β-mercapto-ethanol and proteaseinhibitors (Complete, Roche). Lysates were first denatured by heatingthem for 10 minutes at 70° C. and then treated with EndoH (1 unit/30 μl,Roche Applied Science) or EndoF (1 unit/30 μl, Roche Applied Science)for 19 hours at 37° C. and analyzed by SDS-PAGE and Western blotting.

Generation of Recombinant GST-Fusion Proteins

pGEX-4T-1 plasmids (Pharmacia) encoding GST fusion proteins wereintroduced in BL21-competent cells (Merck Eurolab) and expression of theGST proteins was induced by 0.1 mM isopropyl β-D-thiogalactopyranoside(IPTG, Promega). Recombinant proteins were released from the bacteria bysonication in a Tris-saline buffer (150 mM NaCl, 10 mM Tris) containinga protease inhibitor cocktail (1 mM EDTA, 14 μg/ml aprotinin, 2 μg/mlpepstatin), 100 μg/ml lysozyme, 5 mM DTT and 0.5% N-laurylsarcosine(sarcosyl) (Frangioni and Neel, 1993). After centrifugation at 12500 rpm(Beckman J2-21M/E) to remove insoluble bacterial debris, TRITON® X-100was added to a final concentration of 1% to neutralize the effects ofthe ionic detergent sarcosyl.

Immunization of a Dromedary and Llama and Analysis of the ImmuneResponse

The immunization of dromedary and llama, the isolation of BACE1-bindersand affinity measurements were done in collaboration with Prof. S.Muyldermans, VUB, Belgium.

With weekly intervals, a dromedary and llama were immunized six timessubcutaneously with 150 μg of pure recombinant human BACE1 mixed withGERBU adjuvant (GERBU Biochemicals). The immunogen used for theimmunization of the dromedary was provided by Dr. S. Masure (Johnson &Johnson Pharmaceutical Research & Development, Beerse, Belgium). Inorder to obtain large amounts of active recombinant BACE1, insect cellswere infected with baculoviruses encoding BACE1 ectodomain (sBACE1) inwhich the four putative N-glycosylation sites were removed bysubstituting the respective Asn codons for Gln codons (Bruinzeel et al.,2002). The lack of glycosylation made it possible to produce a large,homogeneous pool of BACE1.

A llama was immunized with a different source of BACE1. In this case,the antigen was purified from sBACE1-overexpressing HEK293 cells(obtained from Prof. N. Mertens, Protein Service Facility, VIB, UGent)and, hence, resembled much better native, mature and thus fullyglycosylated BACE1.

Forty-five days after the first injection, anticoagulated blood wascollected. BACE1-specific antibody titers for each IgG subclass wereanalyzed using ELISA. The three individual IgG subclasses were firstpurified from serum based on their differential absorption on Protein Aand Protein G and distinct elution conditions (Conrath et al., 2001a).Solid-phase coated BACE1 protein was incubated with serial dilutions ofthe different IgG subclasses and bound IgGs were subsequently detectedwith a rabbit anti-dromedary IgG antiserum and anti-rabbit IgG-alkalinephosphatase conjugates (Saerens et al., 2004).

Construction of a V_(H)H Gene Fragment Library

Peripheral lymphocytes were isolated from the dromedary/llama sera(LYMPHOPREP®, NYCOMED®) and total RNA was extracted (according toChomczynski and Sacchi, 1987). After RT-PCR with a dN₆ primer, the cDNAobtained was used as template for the amplification of a DNA fragmentspanning the IgG variable domain until the CH2 domain, using primersCALL001 and CALL002 (see Table 4). These primers anneal to the IgGleader sequence and the CH2 exon of the heavy chain of all three IgGsubclasses existing in dromedary, respectively. Using agarose gelextraction, the 600 bp fragment coming from heavy chain-only antibodies(V_(H)H-CH2, without CH1 domain) was separated from the 900 bp fragmentderived from conventional antibodies (V_(H)-CH1-CH2 exons). V_(H)H genefragments were then amplified by PCR on the 600 bp DNA with a pair ofnested primers, AE6 and FR4FOR (see Table 4). AE6 anneals to the V_(H)Hframework-1 and contains a Pst I site, whereas FR4FOR with a Not I siteis complementary to the framework-4. The different V_(H)H fragments wereligated into a pHEN4 phagemid vector and transformed into E. coli TG1cells to create a library of 4×10⁷ transformants. Colony PCR screeningshowed that approximately 90% of the colonies were transformed with aphagemid vector containing an insert with the size expected for a V_(H)Hfragment.

TABLE 4  Sequences of the different primers used for theV_(H)H gene fragment library construction Primer Sequence (5′ to 3′)SEQ ID NO: CALL001 GTCCTGGCTGCTCTTCTACAAGG 44 CALL002GGTACGTGCTGTTGAACTGTTCC 45 AE6 GATGTGCAGCTGCAGGAGTCTGGAGGAGG 46 FR4FORGGACTAGTGCGGCCGCTGCAGACGGTGAC 47 CTGGGT

Selection of BACE1-Specific V_(H)H Fragments

The V_(H)H repertoire was expressed onto the surface of phages afterrescuing the library with M13K07 helper phages. Specific V_(H)Hs againstBACE1 were enriched by three consecutive rounds of in vitro selection, atechnique also known as panning (Smith and Petrenko, 1997). For this,the V_(H)Hs were incubated on a solid phase coated with antigen. Unboundphages were washed away in PBS plus 0.05% TWEEN® 20 and bound phageswere eluted with 100 mM triethylamine (pH 10.0). Eluted phage particleswere immediately neutralized with 1 M Tris-HCl (pH 7.5) and used tore-infect exponentially growing E. coli TG1 cells. After the second andthird round of selection, individual colonies were randomly picked.

Enzyme-Linked Immunosorbent Assay (ELISA)

Expression of the selected V_(H)H was induced with 1 mM IPTG. Therecombinant soluble C-terminally Hemagglutinin (HA)-tagged V_(H)Hs (thegene encoding the HA-epitope is included in the pHEN4 phagemid vector)were extracted from the periplasm by an osmotic shock (200 mM Tris-HClpH 8.0, 250 mM sucrose, 0.5 mM EDTA) (Skerra and Pluckthun, 1988) andtested for their capacity to recognize their antigen in ELISA tests.Maxisorb 96-well plates (Nunc) were coated overnight with BACE1 protein(100 μl of 1 μg/ml in PBS) at 4° C. Residual binding sites were blockedfor two hours at room temperature with 1% (w/v) casein dissolved in PBS.This antigen-coated solid phase was then incubated with the differentperiplasmic extracts for one hour at room temperature. After washing,the solid phase was successively incubated with mouse anti-HA, alkalinephosphatase-conjugated anti-mouse (Sigma) and 2 mg/ml p-nitrophenylphosphate (Sigma). Signals were analyzed at 410 nm.

Expression and Purification of V_(H)Hs

The V_(H)H genes of the clones scoring positive in ELISA were subclonedinto the expression vector pHEN6, using Pst I and BstE II. Thereby, theHA-epitope tag at the C-terminus of the V_(H)H molecules was replaced bya his6-tag. E. coli WK6 cells were transformed with the pHEN6 plasmidsand expression of the recombinant soluble V_(H)H proteins was induced byIPTG (Saerens et al., 2004). Soluble V_(H)H molecules were extractedfrom the bacteria using an osmotic shock (Skerra and Pluckthun, 1988).The his-tagged recombinant proteins were then captured on anickel-nitrilotriacetic acid superflow Sepharose column (QIAGEN®),eluted with an acetate buffer (pH 4.7), and additionally purified bysize-exclusion chromatography.

BIAcore Measurements

The kinetic constants and affinity of the V_(H)H-antigen interactionswere determined by surface plasmon resonance technology on a Biacore3000 (Biacore AB). Purified V_(H)H molecules, in a concentration rangeof 0-500 nM in Hepes Buffered Saline pH 7.0 or citrate buffer pH 5.0,were injected at 30 μl/minute onto BACE1 (500 resonance units),immobilized on a CM5 chip (according to De Genst et al., 2005). Thekinetic and equilibrium constants (k_(on), k_(off) and K_(D)) weredetermined with the BIAevaluation v3.1 software (Biacore AB).

In Vitro FRET-Based Analysis of β-Secretase Activity

To determine whether the V_(H)Hs affect BACE1 activity, an in vitroBACE1 FRET assay kit was used (Panvera P2985). This assay uses asynthetic BACE1 substrate that emits light upon cleavage. The amount oftotal fluorescence is linearly related to the cleavage rate of thesubstrate and hence to β-secretase activity. Reaction mixturescontaining 20 nM of recombinant BACE1 enzyme and 250 nM of syntheticsubstrate were incubated with an excess of each V_(H)H (2.2 μM) or theBACE1 inhibitor STA-200 (Enzyme System Products, 2.2 μM) in 50 mM sodiumacetate, pH 4.5 at room temperature, protected from light. After twohours, fluorescence was measured at 595 nm using VICTOR 1420 multilabelcounter (Perkin Elmer Life Sciences). For each V_(H)H, the backgroundsignal, emitted by a mix containing V_(H)H and substrate but no enzyme,was subtracted from the signal measured for the mix containing V_(H)H,substrate and BACE1. As an alternative source of β-secretase activity,microsomal membranes were generated from HeLa cells ectopicallyexpressing BACE1 as described hereinbefore. The resulting microsomalpellet was resuspended in 50 mM sodium acetate, pH 4.5. Fifty μg ofmicrosomal proteins were mixed with 250 nM of the synthetic BACE1substrate and 2.2 μM of V_(H)H or STA-200. The enzymatic reaction andanalysis were performed as before except that reactions were gentlymixed every ten minutes during the two-hour incubation.

In another approach, FRET peptide substrateMCA-S-E-V-N-L-D-A-E-F—R-K(Dnp)-R—R-R-R-NH2 (SEQ ID NO:48) wassynthesized by Ana Spec Inc. (San Jose, Calif., USA). Enzyme human BACE1(1-460): IgGFc was purified from HEK293 cells according to the protocoldescribed previously (Yang et al., 2004). For the reaction, enzyme wasdiluted in reaction buffer (50 mM Ammonium Acetate, pH 4.6, 3% BSA, 0.7%TRITON® X-100) at a concentration of 1 μg/ml, and substrate was dilutedin reaction buffer at a concentration of 125 μM. Twenty μl V_(H)H(diluted in reaction buffer) were mixed with 30 μl enzyme dilution and50 μl substrate dilution in 96-well black polystyrene plates (Costar).The plates were read immediately for baseline signal with Envision (355nm excitation, 430 nm emission, 1 second/well), followed by incubationovernight in the dark at room temperature. The plates were read thefollowing morning using the same reader protocol; the FRET signal(-baseline signal) was used as the readout of enzyme activity in eachreaction.

Co-Precipitation of Human BACE1 with his-Tagged V_(H)Hs Using Ni-Beads

BACE1-overexpressing COS cells were lysed in PBS containing 1% TRITON®X-100 and protease inhibitors (1 μg/ml pepstatin, 14 μg/ml aprotinin,0.5 mM PEFABLOC®). One hundred μg of this protein extract were incubatedovernight at 4° C. with 2 μg of his-tagged V_(H)H proteins and Ni-PDCbeads (Affiland) in binding buffer (342 mM NaCl, 16.2 mM Na₂HPO₄, 6.7 mMKCl, 3.7 mM KH₂PO₄ with 1% TRITON® X-100) with the same proteaseinhibitors as used for cell lysis. The precipitates were washed inbinding buffer supplemented with 10 mM imidazole, to reduce unspecificinteractions, eluted using 300 μM imidazole and resolved by SDS-PAGE.BACE1 was visualized by Western blotting using a polyclonal rabbitanti-BACE1 antibody (ProSci Inc).

Phage Libraries Panning with Biotin-Labeled Antigen

Pannings of V_(H)H phage library were performed with biotin-streptavidinsystem. Purified BACE1 ectodomain protein was labeled withSulfo-NHS-SS-Biotin (Pierce) according to the manufacturer's protocol.V_(H)H libraries were rescued with M13K07 helper phage to generatephages. For panning, 10¹¹ phages were blocked with 1% BSA in 400 μlpanning buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.05% TWEEN® 20for 30 minutes at RT. One hundred μl biotinylated BACE1 was added tophage to a final concentration at 200 nM. Phage and biotinylated BACE1were incubated for one hour at RT with rotation. Meanwhile, 40 μlimmobilized streptavidin (Pierce) were blocked in 1% BSA. After one-hourincubation, pre-blocked immobilized streptavidin were added tophage-biotinylated BACE1 solution and incubated for 40 minutes at RTwith rotation. After incubation, immobilized streptavidin were spinneddown by centrifugation at 3000 rpm for one minute, the supernatantcontaining unbound phage were discarded. The immobilized streptavidinwere washed five times with 1 ml panning buffer; each wash lasted fiveminutes with rotation. After wash, 50 mM DTT were added to immobilizedstreptavidin and incubated for 40 minutes at RT with rotation. Theimmobilized streptavidin were spinned down and the supernatantscontaining eluted phages were used to re-infect E. coli TG1 cells forthe next round of phage panning After three rounds of consecutivepanning, recovered phages were used to infect E. coli TG1 cells andplated out at 10⁻⁴ to 10⁻⁶ dilution, and single colonies were picked forfurther analysis.

Phage ELISA Screening

To generate phage particles with V_(H)Hs displayed on the surface forELISA screening, single colonies from phage panning were inoculated in 2ml 2×TY medium supplemented with 50 μg/ml ampicillin and 1% glucose in24-well plates at 37° C. for eight hours with shaking at 220 rpm. Afteran eight-hour incubation, 5×10⁸ pfu M13K07 helper phages were added toinfect each well of bacterials. Infected bacterials were grown at 37° C.overnight with shaking at 220 rpm. The next morning, bacterials werespinned down by centrifugation at 3000 rpm for 20 minutes; supernatantscontaining phage particles were transferred into 24-well plates. Twentypercent PEG 6000/2.5 M NaCl were added to the supernatants using ⅙volume to precipitate phage particles at 4° C. for 30 minutes. Phageparticles were later retrieved by centrifugation at 3000 rpm for 30minutes, and pellets were resuspended in 100 μl PBS.

For ELISA assay of phage particles, BACE1 ectodomain protein was coatedon 48 wells of each 96-well microtiter plate at 100 ng/well at 4° C.overnight, the non-coated wells were used for control. The next morning,microtiter plates were blocked with 3% mild for one hour at RT. Afterblocking, 100 μl phage particles were added to each coated andnon-coated well, and were incubated for two hours at RT. Plates werethen washed five times with washing buffer (PBS, 0.05% TWEEN® 20). Afterwash, HRP-conjugated anti-M13 antibody (Amersham) was added to each wellusing 1:3000 dilution in 3% milk and incubated for one hour. Plates werethen washed five times with washing buffer. After wash, developingsubstrate 0.02 mg/ml ABTS (Sigma) supplemented with 0.3% H₂O₂ (Sigma)were added to microtiter plates and incubated for 30 minutes at RT.Plates were read at OD405 nm with an ELISA reader.

Periplasmic Extract ELISA Screening

Expression vector pHEN4 containing a PelB (Pectate lysase) signalsequence before the V_(H)H cDNAs, thus V_(H)Hs are exported to theperiplasmic space after expressed in bacterial system. To generateperiplasmic extract containing V_(H)H proteins for ELISA test, singlecolonies from phage panning were inoculated in 1 ml Terrific Broth (TB)medium supplemented with 100 μg/ml ampicillin in 24-well plates at 37°C. with shaking at 220 rpm. When OD₆₀₀ reached 0.6, 1 mM IPTG was addedto the culture to induce the expression of V_(H)H proteins. Bacterialswere further incubated for 15 hours at 28° C. for protein expression.After the incubation, bacterials were harvested by centrifugation at3000 rpm for 20 minutes, cell pellets were dissolved in TES solution (20mM Tris-HCl pH 7.4, 1 mM EDTA, 250 mM sucrose) and incubated on ice for30 minutes. The osmotic shock was given by adding 1.5× volume TES/4 tothe bacterials and incubated on ice for 45 minutes. Supernatantscontaining V_(H)H proteins were collected by centrifugation at 300 rpmfor 20 minutes and further used for ELISA. The ELISA assays followed thesame protocol described above for phage ELISA. To detect V_(H)H proteinsgenerated with a C-terminal HA tag, anti-HA monoclonal antibody(Amersham) was used as primary antibody and alkaline phosphataseconjugated goat anti-mouse antibody (Amersham) were used as secondaryantibody. ELISA plates were developed with p-Nitrophenyl-phosphate(PNPP) substrate (Sigma) and read at OD 405 nm with an ELISA reader.

Adeno-Associated Virus (AAV) Construction and Preparation

For AAV generation, a standard method was followed (Levites et al.,2006). Briefly, the cDNA of V_(H)H Nb_B9, fused with a signal peptidefrom BACE1 at its N-terminal and a Myc-tag at its C-terminal, wasconstructed into an AAV vector containing a hybridcytomegalovirus/chicken β-actin promoter and a woodchuckpost-transcriptional regulatory element. AAVs were generated by plasmidtransfection with helper plasmids in HEK293T cells. Forty-eight hoursafter transfection, the cells were harvested and lysed in the presenceof 0.5% sodium deoxycholate and 50 U/ml Benzonase (Sigma) by freezethawing, and the virus was isolated using a discontinuous iodixanolgradient purified on a HITRAP® HQ column (Amersham Biosciences). Thegenomic titer of virus was determined by quantitative PCR.

Mice

All animal experiments were in compliance with protocols approved by thelocal Animal Care and Use Committees. Dutch-mutant APP transgenic mice(C57BL/6J-TgN(Thy-APP_(E693D))) were kindly provided by the laboratoryof Mathias Jucker, University of Tubingen, Germany.

Stereotaxic Injections

In the first series of experiments, AAV vectors expressing V_(H)H Nb_B9and GFP (negative control), and V_(H)H Nb_B24 (negative control) wereadministrated directly into the hippocampus of three-month-oldDutch-mutant APP transgenic mice. Mice were anesthetized with avertinand placed in a stereotaxic apparatus. AAV preparations were injectedbilaterally (2 μl per site) into the CA3 region of the hippocampus (−2.0mm antero-posterior from bregma, +/−2.3 mm medio-lateral from bregma,and 1.7 mm below dura). Mice were then individually housed and allowedto recover from surgery. Their brains were processed for analyses fiveweeks after treatment.

Neonatal Injections

The procedure was described previously (Levites et al., 2006). Briefly,postnatal day 0 (P0) pups were cryoanesthetized on ice for five minutes.AAV preparations (2 μl) were injected intracerebroventricularly intoboth hemispheres using a 10 ml Hamilton syringe with a 30 gauge needle.The pups were then placed on a heating pad with their original nestingmaterials for three to five minutes and returned to their mother forfurther recovery. Their brains were processed for analyses three monthsafter injection.

Tissue Preparation and Biochemical Analysis of Aβ

To analyze Aβ, the hippocampus (from stereotaxic injections) and thewhole brains (from neonatal injections) were homogenized in TissueProtein Extraction reagent (Pierce) supplemented with COMPLETE™ proteaseinhibitor and phosphatase inhibitor tablets (Roche Applied Science). Thehomogenized samples were centrifuged at 4° C. for one hour at 100,000×g,and the supernatant was used for immunoblot analysis and for Aβ ELISAmeasurements using ELISA kits (The Genetics Company).

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What is claimed is:
 1. An isolated single domain antibody, devoid of alight chain, wherein the single domain antibody specifically binds betasecretase (BACE1) and is able to inhibit cleavage of beta amyloidprecursor protein (APP) as determined in a cellular assay.
 2. Theisolated single domain antibody of claim 1, wherein the single domainantibody is derived from camelids.
 3. A medicament comprising: theisolated single domain antibody of claim 1; and at least onepharmaceutically acceptable carrier.
 4. A medicament comprising: theisolated single domain antibody of claim 2; and at least onepharmaceutically acceptable carrier.
 5. A method of treating a subjectsuffering from Alzheimer's disease, the method comprising: administeringthe single domain antibody of claim 1 to the subject, so as to treat thesubject.
 6. A method of treating a subject suffering from Alzheimer'sdisease, the method comprising: administering the single domain antibodyof claim 2 to the subject, so as to treat the subject.